ANALYSIS OF CELL CYCLE
Miriam Capri and Daniela Barbieri
Dept. Biomedical Sciences, Sect. General Pathology,
Via Campi, 287, University of Modena, 41100 Modena, Italy
1. INTRODUCTION
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Cell cycle and apoptosis are very important functional parameters to assess the cellular metabolism, physiology and pathology. Several techniques have been developed to quantitate these parameters utilizing the differential staining of fluorescent dyes. We are describing four different flow cytometric methods, two for the discrimination of cell cycle phases (A and B) and two for the simultaneous assessment of cell cycle and apoptosis (C and D).
A) Bromodeoxyuridine/Propidium Iodide
The classical method for the analysis of cell cycle distribution is the flow cytometric measurement of DNA content which can simultaneously determine the incorporation of Bromodeoxyuridine (BrdU). The procedure requires that DNA is partially denatured to expose incorporated BrdU to a specific antibody. Denaturation is necessary because antibodies developed so far bind only to BrdU in single-strand DNA. The remaining undenatured DNA is then stained with Propidium Iodide (PI). Green fluorescence from the fluorescein-conjugated antibody is a measure of BrdU incorporation. Red fluorescence from the PI is a measure of DNA. The protocol described here uses high-molarity HCl for the denaturation of DNA. Furthermore, this method may be utilized either for unfixed or for fixed cells in suspension.
B) Cyclins/Propidium Iodide
Cyclins are key components of the cell cycle progression machinery. In particular, the expression of cyclins D, E, A and B1 provides new cell cycle landmarks that can be used to subdivide cell cycle into several distinct subcompartments. In this procedure cyclins expression is detectable using specific monoclonal antibodies (mAbs), and is analysed in respect to DNA content.
Generally, the peak of expression of cyclin D1 can be detected in early G1, the peak of cyclin E is typical of G1/S transition, the peak of cyclin A can be detected during G2/M phases and cyclin B1 is typical of late G2/M. Using this method, compared to the above mentioned protocol, it is possible to distinguish G0 from G1 and G2 from M phases. However, it is necessary to keep in mind that not all cell types behave in the same manner (for example, cyclin D1 is detectable not only in G0/G1 but also in G2/M, even if in a very few cell types).
C) TUNEL/Propidium Iodide
One of the most used protocol for the determination of apoptosis in the different phases of cell cycle is the enzymatic in situ labeling of apoptosis-induced DNA strand breaks (TUNEL). Terminal deoxynucleotidyl transferase (TdT) have been used for the incorporation of fluorescein-labeled nucleotides to DNA strands breaks in situ. DNA content is revealed by red fluorescence from PI. In order to have more details, see the Chapters related to TUNEL technique.
D) F-Actin/Propidium Iodide
The analysis of apoptotic cells and estimation of their cell cycle specificity is also possible using a recent method. This is based on identification of apoptotic cells which have modified their cytoskleton and their DNA content. In specific, paraformaldehyde (PFA) fixation followed by staining of F-actin with fluorescein-conjugated phalloidin and of DNA with PI, are used. Furthermore, this procedure may be utilized also for adherent cells.
2. PROTOCOLS
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A) BrdU/PI PROTOCOL
A.2.1 Materials
BrdU (A2), washing buffer (A1), HCl 4 M, Borax buffer (A1), anti-BrdU antibody (A2), goat-anti-mouse-FITC antibody (A2), PI buffer (A1).
A.2.2. Methodology
1. Cells (1x106/mL) are incubated with BrdU 10 mM at final concentration, for 30 min at 37 °C in controlled atmosphere.
2.Wash twice at 500 g for 1 min using the washing buffer.
3. Resuspend in 0.5 mL of washing buffer and 0.5 mL of HCl 4 M.
4. Mix accurately and incubate for 30 min at room temperature.
5. Wash once as in step 2.
6. Resuspend in 1 mL of Borax buffer.
7. As in step 5.
8. Resuspend in 200 mL of washing buffer and label with 5 mL of mAb ant-BrdU.
9. Incubate for 1 hour at 4 °C in the dark.
10. As in step 5.
11. Resuspend in 200 mL of washing buffer and label with 4 mL of goat-anti-mouse FITC-conjugated antibody.
12. Incubate for 30 min at 4 °C in the dark.
13. As in step 5.
14. Resuspend in 200 mL of washing buffer and 200 mL of PI buffer.
15. Incubate for 15-30 min at 4 °C in the dark.
16. Analyse with flow cytometer equipped with a 488 nm argon laser.
A.3. COMMENTARY
A.3.1 Background information
In this procedure fixed cells by 4% PFA in Phosphate Buffer Saline (PBS) can be utilized. In this case to wash cells once in PBS before to start at step 1 is necessary.
Moreover, both direct and indirect immunofluorescence can be used. The BrdU incorporation is more evident using the indirect method.
A.3.2 Anticipated results
It is recommanded to perform each experiment using a negative and a positive control sample. The negative sample, in order to have a correct setting of instrument, is assessed following all the steps except step 8. The positive sample, in order to make sure that the method works, is assessed by using a proliferating cell line (such as U937, K562, MOLT4, etc.) following all steps.
A.3.3 Time considerations
The protocol is simply but it require a quite long time. Indeed, for few samples, more or less 4 hours are required. The duration of the method is obviously depending on the number of samples.
A.3.4 Key references
1. Dolbeare, F., Gratzner, H:, Pallavicini, M., Gray, J.W. 1983. Proc. Natl. Accad. Sci. U.S.A.80: 5573.
B) Cyclins/PI protocol
B.2.1 Materials
PFA (A2), Triton X-100 (A2), PBS, mouse serum (A2), mAbs anti-cyclins (A2), goat-anti-mouse-FITC (A2), PI (A2), GM+EDTA buffer (A1).
B.2.2. Methodology
FIXATION
In the case of E, A and B1 cyclins, cells (2x106/mL) are fixed by 70% ethanol:
a) put all the reagents in ice;
b) count and centrifuge cells at 375 g for 5 min;
c) resuspend accurately the pellet in 1mL GM+EDTA buffer ;
d) add gently 3 mL of 96% ethanol vortexing samples and working in ice;
e) store the fixed samples at 4°C.
In the case of D cyclins, cells are fixed by 1% methanol-free formaldehyde:
a) put all the reagents in ice;
b) count and centrifuge cells at 375 g for 5 min.;
c) resuspend gently the pellet in 1 mL of 1% formaldheyde in PBS for 15 min in ice;
d) wash in ice-cold PBS as in step b;
e) add 1 mL of 70% ethanol;
f) store the fixed samples at 4°C.
STAINING
1. Wash with ice-cold PBS.
2. Resuspend the pellet in 1 mL of 0.25% Triton X-100 in PBS, vortexing samples and working in ice (for 5 min in the case of D, A and B1 cyclins, for 10-20 min in the case of E cyclin).
3. Wash in ice-cold PBS as in step b.
4. Incubate cells with 150 mL of mAb anti-cyclin (diluited at the concentration of 2.5 mg/mL in PBS + 1% of normal goat serum) overnight at 4°C.
5. As in step 3.
6. Incubate for 1 hour with 150 mL secondary mAb goat-anti-mouse-FITC (1.3 mg/mL) diluited 1:50 in PBS + 1% normal goat serum.
7. As in step 3.
8. Incubate for 4 hours at room temperature and in the dark with 1 mL of 2.5 mg/mL PI in PBS (+ 12.5 mL of 1 mg/mL RNAsi) or incubate overnight at 4°C with 0.8 mg/mL PI in PBS (+ 12.5 mL of 1 mg/mL RNAsi).
9. Analyse with flow cytometer equipped with a 488 nm argon laser.
B.3. COMMENTARY
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B.3.1 Background information
The critical steps in the methodology are cell fixation, permeabilization and the concentrations of anti-cyclin mAbs. For most cyclins optimal fixation is 70% ethanol. This treatment preserves cyclins, lowering the background, non-specific cell fluorescence and resulting in an improved signal-to-noise ratio of the cyclin specific fluorescence. Detection of D cyclin, however, requires fixation in formaldehyde. As far as anti-cyclin mAb concentration is concerned, 2.5 mg/mL is optimal for most cells. Anyway, to test the best concentration for each experimental model, is recommended.
B.3.2 Anticipated results
In this procedure, a negative control sample, which contains only the secondary FITC-mAb, is necessary.
B.3.3 Time considerations
The protocol require around 2 hours before the overnight incubation and 5 hours after.
B.3.4 Key references
1. Darzynkiewicz, Z., Gong, J., Juan, G., Ardelt, B., Traganos, F. 1996. Cytometry of cyclin proteins.
Cytometry. 25: 1.
2. Faretta, M., Bergamaschi, D., Ronzoni S., D’Incalci, M., Erba, E. 1997. Diferences in cyclin B1 expression in cell cycle blocked in the G2/M phase after treatment with anti-cancer agent. A new three parametric flow cytometry analysis. Proceedings of the XIV National Italian Meeting of Cytometry.
3. Gong, J., Traganos, F., Darzynkiewicz, Z. 1993. Simultaneous analysis of cell cycle kinetics at two different DNA ploidy levels based on DNA content and cyclin b measurements. Cancer Res. 53: 5096.
4. Gong, J., Li, X., Traganos, F., Darzynkiewicz, Z. 1994. Expression of G1 and G2 cyclins measured in individual cells by multiparameter flow cytometry: a new tool in the analysis of the cell cycle. Cell Prolif. 27: 357.
5. Gong, J., Traganos, F., Darzynkiewicz, Z. 1995. Discrimination of G2 and mitotic cells by flow cytometry based on different expression of cyclins A and B1. Exp. Cell Res. 220: 226.
6. Widrow, R.J., Rabinovitch, P.S., Cho, K., Laird, C.H. 1997. Separation of cells at different times within G2 and mitosis by cyclin B1 flow cytometry. Cytometry 27: 250.
C) TUNEL/PI protocol
C.2.1 Materials
formaldehyde (A2), ethanol, reaction mixture (A1), TdT buffer (A1), Bio-16-dUTP (A2), TdT enzyme (A2), staining buffer (A1), SSC buffer (A1), BLOTTO (A2), Avidin-FITC (A2), Triton X-100 (A2), PI (A2), DNAase buffer (A1).
C.2.2. Methodology
1. Cells (5x105 - 1x106 cell/sample) are washed twice at 260 g for 5 min using 2 mL of PBS (pH 7.2).
2. Fix by 1 mL of 1% formaldehyde in PBS, on ice for 15 min.
3. Wash once as in step 1.
4. Resuspend in 1 mL of ice-cold ethanol 70% (at this point to store the samples at -20°C for 18 hours or overnight is possible).
5. As in step 3.
6. Resuspend in 50 mL (for each sample) of the reaction mixture, which is prepared during the last spin down.
7. Incubate for 30 min at 37°C water bath.
8. As in step 3.
9. Resuspend in 100 mL (for each sample) of the reaction staining buffer, which is prepared during the last spin down.
10. Incubate for 30 min at room temperature in the dark.
11. As in step 3.
12. Counterstain DNA with 5 mg/mL of PI in PBS.
13. Incubate for 15 min at 4 °C in the dark.
14. Analyse with flow cytometer equipped with a 488 nm argon laser.